Elastin increases biofilm and extracellular matrix production of Aspergillus fumigatus

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h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Bacterial

and

Fungal

Pathogenesis

Elastin

increases

bioﬁlm

and

extracellular

matrix

production

of

Aspergillus

fumigatus

Ildnay

de

Souza

Lima

Brandão

a

_,

_Heloiza

_Maria

_da

_Silva

_{Oliveira-Moraes}

b

_,

Cristina

Maria

de

Souza

Motta

c

_,

_Neiva

_Tinti

_de

_Oliveira

c

_,

Oliane

Maria

Correia

Magalhães

a,∗

a_Universidade_Federal_de_Pernambuco,_Centro_de_Ciências_Biológicas,_Departamento_de_Micologia,_Cidade_{Universitária,}_PE,_Brazil b_Universidade_Federal_de_Pernambuco,_Centro_de_Ciências_Biológicas_Rua_Departamento_de_Micologia,_Cidade_{Universitária,}_PE,_Brazil c_Universidade_Federal_de_Pernambuco,_Centro_de_Ciências_Biológicas,_Departamento_de_Micologia,_Pernambuco,_PE,_Brazil

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r

t

i

c

l

e

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o

Articlehistory:

Received27October2016 Accepted2October2017 Availableonline13February2018 AssociateEditor:WaldirElias

Keywords: Aspergillusfumigatus Bioﬁlm Elastin Extracellularmatrix Hydrophobicity

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Aspergillusfumigatusisanopportunisticsaprobefungusthataccountsfor90%ofcasesof pulmonaryaspergillosisinimmunosuppressedpatientsandisknownforitsangiotropism. Whenitreachestherespiratorytract,A.fumigatusinteractswithstructuralcomponentsand bloodvesselsofthelungs,suchaselastin.Tounderstandtheeffectofthisstructural com-ponent,weexaminedtheeffectofelastinontheproductionanddevelopmentofthebioﬁlm ofA.fumigatus.InRPMIcontaining10mg/mLofelastin,asigniﬁcantincrease(absorbance

p<0.0001;dryweightp<0.0001)intheproductionofbiofilmwasobservedincomparisonto whenRPMIwasusedalone,reachingamaximumgrowthof18.8mg(dryweight)ofbiofilmin 72h.Inaddition,elastinstimulatestheproduction(p=0.0042)ofextracellularmatrix(ECM) anddecreases(p=0.005)thehydrophobicityduringthedevelopmentofthebiofilm.These resultssuggestthatelastinplaysanimportantroleinthegrowthofA.fumigatusandthatit participatesintheformationofthickbiofilm.

licenses/by-nc-nd/4.0/).

Introduction

Aspergillus fumigatus is an opportunistic saprobe fungus that accounts for 90% of pulmonary aspergillosis cases in immunosuppressedpatients.Thisdiseasecanexhibitvarious clinical forms, mainly consisting of allergic

bronchopul-∗ _{Corresponding}_author.

E-mail:olimicomed@yahoo.com.br(O.M.Magalhães).

monaryaspergillosis,aspergilloma,andinvasiveaspergillosis (IA),whichareimportantcausesofmorbidityandmortality rangingfrom70to90%.1,2

In aspergilloma and IA, A. fumigatusbehavesas a mul-ticellularcommunitysurroundedbyanextracellularmatrix (ECM),whichischaracteristicofabioﬁlm3,4_and_may_explain,

togetherwithhistologicalevidence,theresistanceto antifun-galagentswhentheseclinicalformsaretreated.5,6

Thedevelopmentofthisfunguswithinthelungsandthe angiotropism7,8_allow_this_{microorganism}_to_be_in_direct

con-tactwithelastin,oneofthemainstructuralcomponentsofthe

https://doi.org/10.1016/j.bjm.2017.10.004

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lungsandbloodvessels,whichisfundamentalfortheir physi-ology.CorrelationbetweenelastaseproductionbyA.fumigatus

andthedevelopmentofIAhasbeenobserved.9

Ithasrecentlybeen demonstrated theinﬂuenceofhost factorssuchasserumcomponents,asfetuinA,10_and

extracel-lularDNA11_in_the_promotion_of_growth_of_A._fumigatus_bioﬁlm;

however,nostudieshaveinvestigatedtheinfluenceoflung tissueconstituentsonthepromotionofbiofilmdevelopment. Inthisperspective,theaimofthisworkwastodetermine theinfluenceofelastininthegrowthanddevelopmentofthe biofilmofA.fumigatus.

Materials

and

methods

Fungalstrainandgrowthconditions

Basedondataobtainedfrom previousanalysisofvirulence factorssuchasbioﬁlmandgliotoxinproduction,andabilityto causepulmonaryaspergillosisinmice(unpublisheddata),we selectedtwoisolatesofA.fumigatus,URM5992 (environmen-talorigin) andURM6575 (clinicalsample),fromthe Culture Collection University Recife Mycology(URM) ofthe Federal UniversityofPernambuco(UniversidadeFederalde Pernam-buco–UFPE),Recife,Pernambuco(PE),Brazil,wereused.The isolatesweremaintainedat28◦Cinmaltextractagar.

Growthconditionsandinoculumstandardization

A.fumigatusisolatesweregrownonSabourauddextroseagar at37◦Cfor72h.Theconidiawerecollectedbywashingthe surfaceofthe culturewith5mLofphosphatebuffersaline (PBS),pH7.2,supplementedwith0.025%(v/v)Tween20.The inoculumwasadjustedto1×105_cells_in_RPMI₁₆₄₀

(Sigma-AldrichCorporation,USA)andbufferedtopH7.0with0.165M MOPS(Sigma-AldrichCorporation,USA)fortheproductionof bioﬁlmin96-wellplates.12_For_{quantiﬁcation}_of_the_dry_weight,

anotherinoculumwasadjustedto3.75×104_cells/cm2_.10

ProductionofA.fumigatusbioﬁlm

A. fumigatus bioﬁlm were produced in ﬂat-bottom 96-well polystyreneplates.Then,200␮Lofthestandardizedcell sus-pensionofeachA.fumigatusisolatewasaddedseparatelyin MOPS-RPMI1640(Sigma-AldrichCorporation,USA)or MOPS-RPMI 1640containing elastin (RPMI/Elastin) (Sigma-Aldrich Corporation,USA)atconcentrationof10mg/mLforeachtime (24,48,and72h).Plateswereincubatedat37◦C.Foreachtime interval,theculturemediumwasremovedfromthewells,and thecellswerewashedthreetimeswithPBS,pH7.2,toremove allnon-adherentcells.12

Toquantifythedryweightofthebioﬁlm,3mLsuspensions ofeachisolatewere placedseparatelyin6-wellpolystyrene plateswithMOPS-RPMI1640orRPMI/Elastin(10mg/mL), incu-bationtimes,andtemperatureslistedabove.10

Bioﬁlmquantiﬁcation

Biofilm was quantified using the technique developed by O’Tooleand Kolter13 _and _subsequently _modified_by _Mowat

etal.12_The_plates_were_dried,_and₁₀₀_␮L_of_0.5%_(w/v)_crystal

violetsolutionwasaddedfor5min.Thesolutionwasremoved by thorough washing under running water. Bioﬁlms were unstainedbyadding100␮Lof95%ethanoltoeachwellfor 1min.Theethanolwastransferredtoanothermicrotiterplate (96-well),andtheabsorbancewasmeasuredat570nm(A570) usingaVarioskanFlashﬂuorescencemeterwithSkanItTM_2.4.5

REsoftware(ThermoFisherScientiﬁc,USA).

Quantiﬁcationofthebioﬁlmbiomass(dryweight)

Afterthe predeterminedtime, the biofilmwas removedby scrapingandfilteredthroughpaperfilters(Miracloth/22␮m, Merck, Germany), which were then dried to a constant weight.10

QuantiﬁcationoftheECM

ThebiofilmformedinRPMIandRPMI/Elastin(10mg/mL)for 48hat37◦Cwerestainedbytheadditionof100␮Lofa solu-tion of25␮g/mL Alexa Fluor 488 (CAAF; Life Technologies, Germany)inPBS,followedbyincubationfor45minat37◦C andstirringat250rpm.Thebiofilmwaswashedthreetimes withPBS.11_The_{fluorescence}_intensity_was_measured_using

aVarioskanFlashﬂuorescencemeterwithSkanItTM _2.4.5_RE

software (Thermo Fisher Scientiﬁc, USA) at excitation and emission wavelengths of 485nm and 520nm, respectively. CAAFstocksolutionsof5mg/mLwerestoredat−20◦_C_and

thawedimmediatelybeforeuse.

Quantiﬁcationofbioﬁlmhydrophobicity

A microsphere adhesion assay with fluorescent orange sulfate-modifiedlatexmicrospheres(0.806␮m,Sigma-Aldrich Corporation, USA)was usedtotest biofilm hydrophobicity. ThebiofilminRPMIaloneandRPMI/Elastin(10mg/mL)were washed with 0.1M KNO3,pH 6.5,and then mixed withan

equalvolumeofthe microspheresolution(109_/mL).

Subse-quently, the mixture was stirred for 30s and extensively washedwiththesamesolution.3_The_amount_of_{ﬂuorescence}

emittedresultingfromtheadherenceofthemicrospheresto thehyphaewasmeasuredwithaVarioskanFlashﬂuorescence meterwithSkanItTM2.4.5REsoftware(ThermoFisher Scien-tiﬁc,USA)atexcitationandemissionwavelengthsof520nm and540nm,respectively.

Bioﬁlmmicroscopy

For microscopicanalysis, the bioﬁlm was grownon cover-slips(22mm×22mm)inRPMIand RPMI/Elastin(10mg/mL) at37◦Cfor48hin6-wellpolystyreneplates.Thecoverslips wereremoved,andthebioﬁlmwasanalyzed.

To visualize the structure of the biofilm, 100␮L of CalcofluorWhite®(Sigma-AldrichCorporation,USA)and exci-tation/emissionfiltersof346/433nmwere usedtoobtain a bluecolor.TheECMquantificationandhydrophobicityassays wereconductedasdescribedaboveforthequantificationof fluorescence.

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TheimageswereobtainedusingaLeicaDMI4000B ﬂuo-rescencemicroscopeandLeicaMicrosystemsLASAFsoftware (Germany).

Analysisofbioﬁlmstructurebyscanningelectron microscopy(SEM)

TheA.fumigatusbiofilmsforelectronmicroscopywere devel-opedasdescribedinthepreviousbiofilmformationsection, in6-wellpolystyreneplates at37◦C during48h wereused forthisexperiment.FortheSEM,thesampleswereprocessed asdescribedbyGonzález-Ramírezetal.14_Briefly,_the_biofilms

werewashedwithPBSandfixedwith2%Glutaraldehyde (Elec-tronMicroscopySciences®,WashingtonPA,USA)for2h.Then, thebiofilmswerepost-fixedwith1%OsmiumTetroxide (Elec-tronMicroscopySciences®,WashingtonPA,USA)for2h.The bottomsofthe6-polystyreneplateswerecutwithahotpunch, andtheintactbiofilmwasobtained.Thesampleswere dehy-dratedwithethanolat10,20,30,40,50,60,70,80and90%for 10minandwithabsolutealcoholfor20min.Then,thebiofilms wereplacedintoacriticalpointdryerandwerecoatedwith ionizedgoldfor400sat15.0kV.TheSEMimageswereobtained usingaShimadzu-SS550microscopewithtungstenfilament (Kyoto,Japan).

Statisticalanalysisanddatapresentation

All datarepresent themean and standard deviationof six replicatesofeach isolate.Thegraphical representationand statisticalanalysisofexperimentaldatawereperformedby analysisofvariance(ANOVA)and/orTukey’stestformultiple comparisonsusingtheGraphPadPrism5software(GraphPad Software,Inc.,California,USA).pvalueslessthan0.05were consideredstatisticallysigniﬁcant.

Results

Bioﬁlmgrowthinthepresenceofelastin

ThebiofilmformationbyA.fumigatus(URM5992andURM6573) wasassessedinmicroplateassayusingcrystalviolet stain-inganddryweight.Theadditionofelastin(10mg/mL)tothe mediumpromotedsignificantbiofilmgrowth(p<0.0001)inall evaluatedtimepoints(Fig.1AandB).Nodifferenceinbiofilm production was observed between the isolates (p=0.1447). Regardlessofthesource,bothstrainswere abletoproduce biofilm.

As expected, the addition of elastin led to increased biomassafter48h,whencomparedtobiofilminRPMImedium alone(9.4mg),reaching18.8mgat72h.Thesedatastrongly suggesttheinfluenceofelastinonA.fumigatusbiofilmgrowth anddevelopment.

Biofilmstabilitywasassessedbyshearmechanicalforce byseriallypipettingthebiofilmswithPBSduringthe wash-ingprocedure (results notshown).Weobservedduringthe first24hofgrowth,thebiofilmappearedconsistent,butitdid notstronglyadheretotheplates.After48h,thestructurewas stableandadherent.

Morphologically,bundles ofparallelhyphae (Fig. 1E and F) as well as an interlaced arrangement, acting possibly to strengthen and stabilizethe structure asa whole,were observedat48h.Inaddition,weobservedcircular arrange-ments (Fig. 1E and F), indicatingthe possible formationof channelsforaircirculation.

IncreasedofECMprovidedbytheelastin

Elastin (10mg/mL) increasedthe amount ofECM produced byA. fumigatus(p=0.0042)(Fig. 2)when comparedtoRPMI mediumalone.ThisincreasebecomesthebioﬁlmofA. fumiga-tusstrainstobemorecohesive,asshownphotomicrographs (Fig.3AandB).Inaddition, weobservedstrongstainingby CAAFinamorphousmaterialsaroundtheendsofthehyphae (Fig.3CandD).Theseresultsindicatestronglythatpulmonary constituentstimulatesA.fumigatustodevelopevasion mech-anismtohostdefenses.

Modiﬁcationofhydrophobicityofthemyceliumbythe elastin

Wefounddifferenthydrophobicpropertiesinthebioﬁlmof

A. fumigatus. The mycelium grown in the biofilm in RPMI wasmorehydrophobicthanobservedinRPMIsupplemented with elastin(10mg/mL) (Fig. 4movefigure2). The hydropho-bicitywasassessedinhydrophobicityassayofthemycelium surfacewithfluorescentorangesulfate-modifiedlatex micro-spheres(0.806␮m,Sigma-AldrichCorporation,USA).

Inthisassay,alargenumberofmicrospheresadheredto the bioﬁlm grown in RPMIalone (Fig. 5A and B), whilein the presenceof elastin, the hydrophobicity was decreased (p=0.005)(Fig.5CandD).Whatisinterestinginthisdatais thatdifferentthanweexpected,thehydrophobicitydecreased whileECMproductionincreased.

Ourﬁndingrevealedthatthepresenceofhostcomponents, suchaselastin,iscapableofalteringthebehaviorofA. fumi-gatus,asshownhereforhydrophobicity.

Discussion

Bioﬁlms are multicellular communities of microorganisms surrounded by an ECM. Growth in communities confers advantagestofungi,includingtheeaseofcolonizationofthe substrate,protectionfrom environmental aggression, resis-tancetophysicalandchemicalstress,metaboliccooperation, andregulationofgeneexpression.3,6

Severalreportshaveshownthataddingnewcomponents toRPMI1640mediumproducesconditionsthatmimicthehost organismduringinfection.4,11,15

Asfarasweknow,thisisthefirstreportofbiofilm produc-tionbyA.fumigatusonthepresenceofelastin.Inthisstudy, thepresenceofelastinintheRPMImediumledtoincreased biomassin100%after48h,whencomparedtobiofilminRPMI mediumalone.Theincreasebiofilmwhenaddedelastintothe growthmedium,suggeststheinfluenceoflungconstituents on the promotion of biofilm development of A. fumigatus.

Recently,hasbeendemonstratedtheinﬂuenceofotherhost factors,suchasserumcomponents,suchasfetuinA,10 _and

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A

_2.0 ** ** **** **** **** **** **** **** **** **** **** 1.5 1.0 0.5 0.0 Time (h) 0 µm50 0 µm50 0 µm50 0 µm50 Time (h) Dr y w eight (mg) Absorbance (A570nm) 20 15 10 5 0 24h 48h 72h 24h 48h 72h

B

D

F

E

C

Fig.1–InfluenceofelastinonthegrowthofA.fumigatusbiofilm.ThegraphicsrepresentthemeanvalueswithSDsofthe biofilmproductionofbothisolatesofA.fumigatusmeasuredbyabsorbancewith0.5%crystalviolet(A)and(B)thedry weight.Asignificantincreaseinthepresenceof10mg/mLofelastin(graybars)comparedtoRPMIalone(blackbars)was observedforalltimeperiods(**p<0.01;****p<0.0001;ANOVA);(C)A.fumigatusURM6575biofilmgrowninRPMIisshown after48hat37◦Cunderlightinmicroscopyand(D)stainedwithCalcofluorWhite®(E)andinRPMIsupplementedwith 10mg/mLofelastinundermicroscopyand(F)stainedwithCalcofluorWhite®.Anincreasedamountofbiofilmwas producedinthepresenceofelastin(10mg/mL).Circulararrangements(yellowarrow)andparallelbundlesofhyphae(black arrow)areshown.Scalebar,50␮m.

extracellularDNA11_on_promoting_the_growth_of_A._fumigatus

bioﬁlm.

Nodifferencewasobservedinbioﬁlmproductionbetween theisolates(p=0.1447).BothisolatesofA.fumigatuswereable toproducebioﬁlm independentoftheirsubstrate oforigin (clinicalorenvironmental).Thisresultisconsistentwiththose ofGonzález-Ramírezetal.14_who_did_not_observe_difference

indevelopmentandbioﬁlmproductionbetweenclinicaland environmentalisolatesofA.fumigatus.

Weobtained maximumdry weight at72h of18.8mgis morethan twice themaximum value(8.3mg) obtainedfor Seidleretal.4_during_the_same_time_period_of_co-culture_with

bronchial epithelialcells,usingthe A.fumigatusATCC9197 strain. While the bioﬁlm ofA. fumigatus(IFM 49896 strain) demonstrated by Toyotome et al.10 _in _presence _of _serum

protein fetuin A reached an average mass of 30mg. Indi-cating that other host constituents may be necessary for bioﬁlmdevelopmentinA.fumigatus.Inaddition,thebioﬁlm

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150 **** ** ** Fluorescence (485/520 nm) 100 50 URM5992 URM6573 0

Fig.2–Inﬂuenceofelastinonextracellularmatrix(ECM)of

A.fumigatusbioﬁlm.ThemeansandSDsshowthatthe

amountofECMproducedinthepresenceofelastin(gray bars)wassigniﬁcantlygreater(p=0.0042),thanthat producedinRPMIalone(blackbars).Thedifference betweenisolatesofclinical(URM6573)andenvironmental (URM5992)originwashighlysigniﬁcant(p<0.001,ANOVA). **p<0.01;****p<0.0001.

productionvariationobservedinthesestudiesmayberelated to the bioﬁlm-forming capacity of each organism, vary-ing according to the isolates used, as known for group A

Streptococcus.16

The observed increase in biofilm production can be attributedtoseveralfactors,firstlytheelastinbeanabundant proteininlung tissueandblood vesselwalls,likelyserving asanimportantsourceofnutrientsforbiofilmproduction.9

Elastaseactivity mustbehighlighted amongallthe factors supposedly related to pathogenicity in this mold, because not only elastincan serve as a source ofnitrogen for the fungus,butalsoits degradationcouldallowpathogen inva-sion through host tissues.17,18 _This _activity _has _also _been

describedinotherimportantpulmonarypathogens,suchas

Pseudomonasaeruginosa.19

Moreover,the elastincouldbeable topromote modula-tionofgenesinvolvedintheregulationofbiologicalprocesses, whichmayberelatedtotheestablishmentofdeepinfections, likehasbeendemonstratedtoTrichophytonrubrum.17

Previous studies evaluating bioﬁlm growth kinetics observedincreaseforbioﬁlmproducedovertime,3,12_which_is

ingoodagreementwiththeresultsofthepresentstudy. Regarding morphology, the hyphae arrangement in the bioﬁlm of A. fumigatus observed is similar to that found bySeidleretal.,4 _where_{parallel-packed}_hyphae _strengthen

thestructureinonedirectionwhilecrossinghyphaefurther stabilizethe structure.Inaddition,the presenceofcircular

A

AccV Probe Mag WD Det 5um

0 µm 25 0 µm 25 SE 17 x 2000 4.0 15.0kV

AccV Probe Mag WD Det 5um SE 17 x 2000 4.0 15.0kV

B

D

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Fig.3–Enhancedproductionextracellularmatrix(ECM)ofA.fumigatusbiofilm.Photomicrographstakenbyscanning electronmicroscopyanalysisofA.fumigatusURM6575biofilminRPMI(A)andRPMIwithelastin(B)showincreased productionofECMinthepresenceofelastin(yellowarrow).Magnificationof2000×.A.fumigatusURM6575biofilmgrownin RPMIsupplementedwith10mg/mLofelastin(C)underlightmicroscopyandstainedwithconcanavalinA-AlexaFluor488® (D)showgreateramountsofECMattheendsofthehyphae(whitearrow).Scalebar,50␮m.

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2000 _**** ** ** Fluorescence (520/540 nm) 1500 1000 500 0 URM5992 URM6573

Fig.4–InﬂuenceofelastinonthehydrophobicityofA.

fumigatusbioﬁlm.Thehydrophobicityinthepresenceof

elastin(graybars)wassigniﬁcantlylower(p=0.005)than thatinRPMIalone(blackbars).Thedifferencebetweenthe clinical(URM6573)andenvironmental(URM5992)isolates washighlysigniﬁcant(p<0.0001,ANOVA).**p<0.01; ****p<0.0001.

arrangements,alsoobservedbyBeauvaisetal.3_suggests_the

possibleformationofchannelsforaircirculation,whichcould betheoriginoftheoxidationmechanismsresponsibleforthe productionofmelanin,aknownconstituentoftheECM.3,20

As expected, the increase of bioﬁlm was followed by increaseamountofECM,thisresultisconsistentwiththose ofSeidleretal.4_who_observed_the_increase_in_the_amount_of

ECMisrelatedtothegreateramountofbioﬁlm.

Thismatrix is anessential component forthe develop-mentofbioﬁlmbecauseitgluestogetherthehyphaeandﬁxes themtothesurface.3_In_addition,_it_provides_protection_against

externalfactors,suchastheactionofthehostimmunecells andantifungalagents.20–22

Inthisstudy,weusedCAAFtostainthepolysaccharides and SEM to analyze the ECM. In agreementwith previous studies,3,4,10_we_found_the_ECM_as_a_matrix_diffused_between

thehyphaeand surroundsthem,whereitapparently glues togetherthehyphalthreadsofthenetwork.3

Consistentwithearlyobservations,3,4,11_the_ECM_was_also

moreevidentendingﬂowtubes,suggestingitnotonlycovers andprotectsthestructureasawholebutalsoconnectsthe endsofthehyphaetoeachother.11

Thepresenceofcarbohydrate,evidenced byCAAF stain of␣-mannopyranosyl and␣-glucopyranosyl residuesofthe polysaccharides,suggestsitsavailabilityintheECMisaresult ofneedtoprovidenutritiontothehyphaethatmakeupthe bioﬁlmstructure,especiallythosethataremostdistantfrom thenutrientsource.12

Ourﬁndingsconsistofamulticellularcomplexstructure andhighlyorganized, withpolysaccharidesinthecell wall andsurroundingthe hyphaeinthebioﬁlmlikereportedby earlystudies.3,4,12,22

This is the first study to observe an increase in ECM productioninA.fumigatusbiofilminthepresenceofa pul-monaryandbloodvesselsconstituent,theelastin.Thisfinding

suggeststhedevelopmentofbioﬁlmofA.fumigatusisstrongly inﬂuencedbyfactorspresentinthehostbecausetheECMis alsopresentinaspergillomaandprovidesgreaterstabilityto thehyphaenetwork.20_In_addition,_it_is_hypothesized_that_ECM

playsasigniﬁcantroleinantifungalresistancebyadsorbing antifungaldrugmoleculesandpreventingtheirdiffusion.4,23

Apossibleexplanationforthismightbethegreater avail-abilityofnutrients,whichprobablyledtohighergrowthwith increasedproductionofECM.Previouslywas demonstrated thefundamentalroleofelastaseontissueinvasion, suggest-ing A.fumigatusopens breachesinthepulmonarybarriers, secretingtheseproteases,whicharerelatedtopathogenicity thisfungus.24

Anotherpossibleexplanationforthisisthattheincrease inECMproductionobservedinthisstudymayberelatedto thestimulationofthepulmonaryenvironment,whichcauses

A. fumigatustodevelop strategiestosurvivethe host.This matrix contains toxins,suchasgliotoxin, whichiscapable of inducingapoptosis inmacrophages, polymorphonuclear leukocytes and dendriticcells,aswellasmelanin, the pig-mentthatprotectsthefungusagainstoxidativestress.3,15,25,26

Thus,assuggested,thelungenvironmentappearstoselect theisolatesthatarebestadapted.27,28

Intermsofhydrophobicity,weusedanassaywith sulfate-modiﬁedlatexmicrospheres,becausetheyhavealowdensity ofnegativecharges, andmorethan 90% oftheirsurfaceis availableforhydrophobicinteractions.29

Adhesionistheﬁrststepincolonizationofasubstrateby afungus,whichoccursthroughvariousinteractionsbetween conidiaandthesubstratesurface.30_Despite_the_importance

of the adhesion, little isknown about this process.31

Sev-eral studieshavefocusedprimarilyon thefungal cellwall, emphasizing proteins, especially the hydrophobins, which are responsible forthe high hydrophobicity ofconidia and hyphaewalls.Theseproteinsstabilizetheadhesionofspores tohydrophobicsurfaces,bothnaturalandartiﬁcial,possibly generatingmorphogeneticsignals.30,32_.

In A. fumigatus have been demonstrated hydrophobins are responsible for the hydrophobic characteristic of the ECM.3,33,34 _Bruns _et _al.15 _and _Gibbons _et _al.35 _showed

up-regulationofhydrophobinsgenesofA.fumigatusinbioﬁlm condition,suggestingthatseveralhydrophobinsareexpressed intheA.fumigatusECM.

Thisistheﬁrststudytoreportachangeinthe hydropho-bicityofA.fumigatusbioﬁlmwhenelastin,afactorpresentin thehost,isaddedtogrowthmedium.Environmental condi-tionssuchastemperature,nutrientsupply,andhumidity,36_as

wellastheculturingconditions,i.e.,solidorliquidmediaand bioﬁlmconditions,canaffecthydrophobicity.3,37,38

Contrary to expectations, this study ﬁnds a signiﬁcant decrease on hydrophobicitywhen elastinisaddedtoRPMI medium,eventhoughtheirpresencehascausedanincrease intheECM.

This inconsistency may be due to the role played by hydrophobins in completing the life cycle of these fungi, causingthesurfacetobehydrophobicandresistantto humid-ity,therebyfacilitatingthedispersionofsporesthroughthe air.39,40_Thus,_after_established_a_community_in_the_lung

envi-ronment,A.fumigatus,mostlikely,doesnotneedtoexpend energyforproductionofdispersingproteins.

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A

B

D

0µm 50 0µm 50 0µm 50 0µm 50

C

Fig.5–DecreasedhydrophobicityofA.fumigatusbiofilm.A.fumigatusURM5992(environmentalorigin)biofilmafter48hat 37◦C.(AandB)BiofilmGrowninRPMIwithoutelastinunderlightmicroscopyandstainedwithlatexbeads,respectively.(C andD)biofilmgrowninRPMIwithelastin–10mg/mLunderlightmicroscopyandstainedwithlatexbeads,respectively. Scalebar,50␮m.

However,otherroleshavebeenattributedtohydrophobins, asdemonstratedbyRodAp,whichhamperimmune recogni-tion;anditsabsenceisassociatedwithareductionofvirulence inthisfungus.41–43_These_ﬁndings_reinforce_the_need_for

fur-therstudiestoelucidatethebiologicalrolesofhydrophobins, especially in the context of the host-parasite relationship becauseseveralhostfactorsmayacttogether,inadditionto elastin,toinducetheexpressionoftheseproteinsinvivo.

Inconclusion,thisstudydemonstratedthatelastin influ-ences biofilm development of A. fumigatus. The results showedthattheECMproductionwasstronglyincreasedwhile the hydrophobicity of the biofilm decreased. The present studyconfirmspreviousfindingsandcontributesadditional evidencethatsuggestspulmonaryconstituentscanalso influ-encebiofilmdevelopmentofA.fumigatus.

However,additionalexperimentsshouldbenecessaryto allowforincreasedunderstanding of the role ofhost con-stituentsfordevelopmentofA.fumigatusbioﬁlm.

Conﬂicts

of

interest

Theauthorsdeclarethattheyhavenocompetinginterests.

Acknowledgements

TheauthorsaregratefultotheNationalCouncilforScientiﬁc andTechnologicalDevelopment,Brazil(ConselhoNacionalde DesenvolvimentoCientíﬁcoeTecnológico–CNPq)forsupport.

In additiontothankingthe Dr.Dijanah CotaMachado and Dr.PaulEuzébiooftheDepartmentofPhysiologyand Depart-mentofBiophysics,respectively,oftheFederalUniversityof Pernambuco,Brazil.

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